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Abstract

Plants are constantly assaulted with abiotic and biotic stresses. They have
developed mechanisms to transduce those stresses into adaptive physiological
responses, including the production of reactive oxygen species as an intermediate
signal, a mechanism they share in common with animal responses to stress. In
animals, extracellular ATP (xATP) is a signal that induces the production of
reactive oxygen species. In this dissertation, we document that xATP can serve as
a signal also in plants by inducing superoxide production via NADPH oxidases.
We also characterize intermediate elements in the ATP signaling pathway,
downstream gene expression changes and the possible regulation of this signal by
apyrases in Arabidopsis thaliana.
ATP- treated Arabidopsis leaves had increased superoxide accumulation.
Inhibitors of the P2 receptors that initiate xATP responses in animals were able to
block ATP-induced superoxide production in Arabidopsis. Mutants missing
NADPH oxidase subunits did not show ATP-induced superoxide accumulation,
indicating that NADPH oxidase activity is responsible for the ATP-induced
superoxide production. A cation channel blocker, a calcium chelator, and a
calmodulin antagonist also blocked this ATP response, implicating increases in
[Ca2+]cyt and the activation of calmodulin as intermediate signaling steps between
xATP and superoxide production. Genes that are induced by various stresses were
up-regulated by xATP, including genes involved in the biosynthesis of jasmonates
and ethylene.
Ectoapyrases are enzymes that hydrolyze extracellular nucleotides.
Arabidopsis apyrases were assessed as possible regulators of the xATP signal, a
role they could carry out directly by quenching the signal and indirectly by
increasing the AMP product that can readily be converted to adenosine, a negative
feedback suppressor of ATP effects in animals, although this has not been
demonstrated in plants to date. Adenosine decreased ATP-induced superoxide
production, and three independent lines overexpressing the apyrase gene, Atapy2
(Arabidopsis thaliana apyrase 2) had reduced superoxide production in response
to ATP treatment. The level of two mRNAs encoding apyrases increased in
response to wounding, but only Atapy2 had increased gene expression when
treated with oligalacturonic acid (OGA). These differences suggest differential
regulation of these similar apyrases. This dissertation provides evidence for a
novel signal transduction pathway in Arabidopsis leading to the induction of
superoxide production and the possible regulation of this pathway by apyrases.